Role Of KLF₄ In The Mechanism Of Pathogenesis Of Epilepsy

Epilepsy is a common neurological disease characterized by transient clinical symptoms or syndromes?such as tonic or spasmodic convulsion?caused by abnormal firing activity of neurons in the brain..The onset of epilepsy mainly results from imbalance between neural excitability and neural inhibition.At present,some mechanisms participate in the pathogenesis of epilepsy,for instance,heredity,neurotransmitters and their receptors,Ion Channels,axons and radial glial cells.Many genes are involved in the mechanisms of pathogenesis of epilepsy,and mutation or aberrant expression of any one of them may cause the onset of epilepsy.Although many genes have been reported to be associated with epilepsy,monogenic epilepsy is rare in patients.In most cases,polygenic and external factors both contribute to epilepsy.With the progression of next generation sequencing and genome editing,the various genes that are causally implicated in epileptogenesis have been found.However,there are not too many studies on transcription factors.Nowadays,some tumor related transcription factors have been reported to play roles in pathogenesis of epilepsy.P53,a well-known transcription factor,has been widelly studied in oncogenesis.Recent studies have found that P53 plays a important role in the development of epilepsy.Increased expression of P53 in hippocampus was found in a variety of epileptic models KLF₄ is an upstream gene and a transcriptional repressor of P53.KLF₄ is involved in many neurobiological processes such as oxidative stress,neuroinflammatory response,neural regeneration,stem cell differentiation and cell apoptosis.Based on the above evidence,we believe that KLF₄ may be involved in the pathogenesis of epilepsy,and the overexpression of KLF4 in hippocampal neurons may produce antiepileptic effects.In the present study,we conducted behavioral pharmacology,electrophysiology,molecular biology and genomics to explore the anti-epileptic effect of KLF₄ and its possible mechanism.In pilot studies,we identiified the effects of antiepileptic drugs VPA and CBZ on behavioral profile of PTZ-induced seizures in rats and the expression of hippocampal KLF4 protein.The results showed that PTZ inhibited KLF4expression,while both VPA and CBZ increased KLF₄ expression.In the mouse PTZ model,we observed the same results as in the rats.Besides,the expression of P53increased in the hippocampus of PTZ mice,and the expression of P53 decreased after VPA treatment.In vitro studies,we primarily cultured mouse hippocampal pyramidal neurons and overexpress the KLF₄ gene in pyramidal neurons through viral infection.Electrophysiological studies showed that the actin potential frequency of pyramidal neurons with KLF₄ overexpression was lower than that of the control group regardless of the presence or absence of PTZ.Then we overexpressed the mouse hippocampal KLF₄ protein through the brain stereotactic microinjection technique,and the behavioral results showed that the overexpression of KLF₄ increased the latency of epilepsy.The Western blot results showed that the increase of KLF₄ expression can directly induce the down-regulation of P53 expression.Meanwhile,we studied the effect of overexpressed KLF₄ in the hippocampus on the c-fos expression in the hippocampus and the piriform cortex through immunohistochemistry and immunofluorescence.The results showed that PTZ significantly increased the expression of c-fos in the hippocampus and the piriform cortex,and overexpressed hippocampal KLF₄ decreased the expression of c-fos in both brain regions.In addition,we predicted the regulation of gaba-related genes by KLF₄ through a chip-req database,and Western Blot proved that the increase of overexpressed KLF₄ actived GAD67 expression,but had no effect on GAD65.Finally,to explore the genes involved in epilepsy,we sequenced the whole genome of 6 children,including 2children with epilepsy and autism,2 children with autism and 2 normal children.The results showed that KLF₄ rs2236599,GABRA6 rs13184586,GABRB2 rs2808536,GABBR1 rs29225 may be epileptic-related SNPs,and GABRG2 rs211037,GABRD rs2229110 and rs28398772,SLC6A13 rs2289954,GABBR2 rs10985765 and rs2304389 may be related to both epilepsy and autism.The above results suggest that the up-regulated KLF₄ expression may participate in the antiepileptic effects of VPA and CBZ.Overexpression of KLF₄ exhibited antiepileptic effects and inhibited increased action potential frequency of hippocampal neurons and increased c-fos in hippocampus and piriform cortex on PTZ-induced seizure in mice.KLF₄ rs2236599,GABRA6 rs13184586,GABRB2 rs2808536,GABBR1 rs29225 may be epileptic-related SNPs,and GABRG2 rs211037,GABRD rs2229110 and rs28398772,SLC6A13 rs2289954,GABBR2 rs10985765 and rs2304389 may be related to both epilepsy and autism.In conclusion,these findings suggest that KLF₄ plays an important role in PTZ-induced seizure by modulation of GABAergic system or p53and might be a new molecular target for epilepsy.